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Atrophy of the alveolar ridge is a problem in prosthetic and esthetic treatment. In the present study, to examine effects of PGE1 on the alveolar bone after tooth extraction, PGE1 was applied to rat incisal sockets utilizing a slow drug release system using PLGA as the drug carrier.Thirty-six male Wistar rats, 10 weeks old, were divided into 3 groups. After all right mandibular incisors were extracted, the sockets were treated in the following manner. The first group was untreated (control group), the second group received a PLGA rod (PLGA group), and the third group was treated with a PLGA rod containing 0.5 mg PGE1 (PGE1 group). Six rats in each group were sacrificed at 4 weeks, and the remaining rats were sacrificed at 8 weeks. For fluorescence labeling, half of the animals were injected with calcein and tetracycline 8 days and 1 day before sacrifice, respectively. After sacrifice, the mandibles were radiologically and histologically examined.In the PGE1 group, the bone volume of the alveolar ridge including the socket was significantly (P.05) greater than in the control and PLGA groups at 4 and 8 weeks. At 4 weeks in the PGE1 group, the mineral apposition rate and number of osteoclasts were higher than in the other groups, whereas these parameters were similar in all groups at 8 weeks.Based on this animal study, it appears that local application has the potential to preserve and/or augment the alveolar ridge after tooth extraction.

Purpose : Optical coherence tomography (OCT) has been investigated as a novel diagnostic imaging tool. The utilisation of this equipment has been evaluated through several studies in the field of dentistry. The aim of this preliminary study was to determine through basic experiments the effectiveness of OCT in implant dentistry. Materials and Methods : To assess detection ability, we captured OCT images of implants in each of the following situations: (1) implants covered with mucosae of various thicknesses that were harvested from the mandibles of pigs; (2) implants installed in the mandibles of pigs; and (3) implants with abutments and crowns fixed with temporary cement. The OCT images were captured before cementation, after cementation, and after removing the excess submucosal cement. results : If the thickness of the mucosa covering the implant body was less than 1 mm, the images of the implants were clearly detected by OCT. In the implants were installed in pigs’ mandibles, it was difficult to capture clear images of the implant and alveolar bone in most of the samples. Remnants of excess cement around the implants were visible in most samples that had a mucosa thickness of less than 3 mm. conclusion: Currently, OCT imaging of implants is limited. Cement remnants at the submucosal area can be detected in some cases, which can be helpful in preventing peri-implant diseases. Still, though there are some restrictions to its application, OCT could have potential as an effective diagnostic instrument in the field of implant dentistry as well. (Imaging Sci Dent 2016; 46: 173-8)

One hypothesis for the mechanism of tooth eruption is that the periodontal ligament fibroblasts generate the eruptive force. To assess the force generated, these fibroblasts were obtained by explant culture of ligament from mandibular premolars of a dog and were cultured in collagen gel matrices. The forces generated by them under isometric conditions were continuously measured for 120 h with a strain gauge. At the same time the number of cells in the gel was counted and the force measured was calculated as the force generated by 104 cells. Shortly after the start of culture, the force per 104 cells increased rapidly; it reached 5.2 × 10−4N at 8 h, and then remained at the same level for about 48 h. Our findings suggest that fibroblasts of the periodontal ligament may generate sufficient force for tooth eruption.

The aim was to assess the influence of connection of the canine teeth to implant-retained long-span fixed dentures on stress in mandibular bone using finite element analysis.Each 3-dimensional model included bone, 6 implants, both natural canines, and superstructures. Each model simulated 1 of 4 prosthetic designs: a tooth/implant-retained 1-piece superstructure (One-piece), 3-piece superstructures with an anterior and 2 posterior segments with unconnected teeth (UnConnect), 3-piece superstructures with the teeth connected to the posterior segments (PostConnect), and 3-piece superstructures with the teeth connected to the anterior segment (AntConnect). A nonlinear elastic modulus was applied to the periodontal ligament (PDL). Maximum intercuspal (IP), canine-protected (CP), and group-function (GF) occlusions were simulated.The maximum stresses in the peri-implant regions of the bone were lower for the One-piece than for the other superstructures. In PostConnect and AntConnect, the maximum stress in the PDL was lower than that in UnConnect, but the stress in the peri-implant bone was comparable in those 3 models. The stresses were lower in the GF model than in the CP model. The stress created in the peri-implant bone was insensitive to the modes of the teeth/implant connection in long-span fixed dentures.Within the limitation of the mechanical analysis, it is suggested that the connection of the canine tooth to the implant-retained long-span superstructures is an acceptable option in partially edentulous patients.

Fibroblast growth factor 4 (FGF-4) plays important roles in bone development during embryogenesis. Human FGF-4 consists of 206 amino acid residues. We produced amino-terminally truncated rhFGF-4, named rhFGF-4(73-206), that increases bone mineral density when systemically administered to mice. In the present study, we examined the effects of rhFGF-4(73-206) in bone after local administration. We injected 1 microg of rhFGF-4(73-206) into tibiae of 10-week-old Sprague-Dawley rats. At days 4 and 7, sets of animals were subjected for tibial bone marrow cell culture in an osteogenic medium. The bone marrow cells from FGF-4-injected tibiae produced more alkaline phosphatase-positive cells and mineralized nodules than those from control tibiae, indicating that local injection of rhFGF-4(73-206) increased the osteoblastic population in the bone marrow. The remaining sets of animals were killed on days 7 and 10. The tibiae were then analyzed with soft X-ray, dual energy X-ray absorptiometry, peripheral quantitative computed tomography, and histomorphometry. The radiographic analyses revealed increases in trabecular bone in the tibiae but no difference in the cortical bone between the rhFGF-4(73-206) group and the control group. High bone turnover and a derived increase of trabecular bone mineral density were demonstrated by histomorphometry in the rhFGF-4(73-206) group. The present results indicate that local injection with rhFGF-4(73-206) elicited an increase in the osteogenic cell population in the tibial bone marrow, which resulted in more trabecular bone.

Peri-implantitis treatment with laser or Titanium bur M. Htet;M. Madi;O. Zakaria;T. Miyahara;X. Wang;Z. Lin;K. Aoki;S. Kasugai; Clinical Oral Implants Research

The purpose of the present study was to compare alpha- and beta-tricalcium phosphate (TCP) as bone graft material for augmenting highly resorbed alveolar ridges. The cranial bones of 15 rabbits were used. Three titanium chambers filled with porous blocks of alpha-TCP, beta-TCP, or blood clots were placed in each slit. The two TCP blocks had similar inner/outer structures and purities. Animals were sacrificed after 2, 4, and 8 weeks. Specimens were embedded in polyester resin as nondecalcified specimens, and evaluated both histologically and histomorphometrically. In both TCP groups, blocks had hardly degraded at 2 weeks while in the alpha-TCP group, the block had notably started degrading after 4 weeks. In the beta-TCP group, degradation began at 4 weeks and this degradation had increased just slightly after 8 weeks. The alpha-TCP block degraded significantly more than the beta-TCP block. Residual alpha-TCP particles surrounded by newly formed bone decreased over time, and both particles and newly formed bone were simultaneously absorbed by osteoclast-like cells. These observations suggest that residual alpha-TCP particles surrounded by newly formed bone may disappear progressively from bone and could be incorporated into the bone remodeling cycle in combination with newly formed bone.

Adhesive restorative systems have expanded the range of possibilities for direct pulp-capping technique, with evidences of clinical success in vital pulp therapy. However, quite few studies have described the direct responses of pulp cells following the application of resinous materials to pulp exposure. To address this issue, effects of exposure to an adhesive resin, 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin on cellular activity were investigated in an established rat dental pulp cell line (RPC-C2A). RPC-C2A cells were cultured on normal plastic plates or the disks prepared from 4-META/MMA-TBB resin (Super Bond CB) in a-MEM containing 10% FBS. After 3, 7 and 14 days, DNA content and alkaline phosphatase (ALP) activity were measured. Total RNA in each group was extracted and RT-PCR analysis was performed. Moreover, the live cell ratio was also evaluated by cytotoxicity assay after treatment with various concentrations of 4-META/MMA-TBB. At day 3, 7 and 14, amount of DNA and ALP activity of the cells on normal plastic plates and the one on the 4-META/MMA-TBB were comparable. Cells of both groups expressed mRNA of type I collagen (Coll), ALP, osteopontin (OPN), osteocalcin (OC), and bone morphogenetic protein (BMP-2). Furthermore, 4-META/MMA-TBB (10(-1)% or less) did not influence dead cell ratio in the confluent state. According to the results of these in vitro studies, exposure to this resinous material would not induce cytotoxic response in the pulp cells.

Platelet-rich plasma (PRP) stimulates tissue healing and centrifugation is the only method for PRP preparation. The purpose of the present study was to develop a method to separate platelets without centrifugation. We used 16 polymers of different chemical characteristics and mixed each polymer with fresh whole blood containing anticoagulant citrate-dextrose. Then, we observed blood cell separation. Focusing on the effective polymers and comparing our method with the conventional centrifugation method, we examined platelet recovery rate and P-selectin expression, which represents platelet activation. Poly-L-glutamic acid (PGA) and poly-L-aspartic acid (PAA) separated platelets efficiently; however, these polymers activated platelets. On the contrary, poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB), which has phosphorylcholine residues mimicking a cellular membrane molecule, separated platelets moderately and the PMB-separated platelets were nearly inactive compared to the separation with the ordinary centrifugation and PGA. Conclusively, the present experiments demonstrated that without centrifugation platelets can be separated from whole blood with some water-soluble polymers, such as PGA, PAA, and PMB, and that PMB has an advantage not to activate platelets. Keywords:: Platelet-rich plasma, separation, aqueous two-phase system, polymer, P-selectin

Prosthetic treatment with dental implant is currently reliable. Since dental implant treatment can yield much satisfaction to patients compared to other treatments, such as bridge or removable denture, number of patients, who desire to be treated with dental implant, is increasing. Cortical bone of female mandible is thin compared to that of male mandible and postmenopausal condition decreases bone density of the cancerous bone. Furthermore, estrogen-deficiency delays implant and bone connection. Thus, it is necessary to treat osteoporotic patients with dental implants carefully compared to usual patients.

The ideal dressing material is bio-inert and keeps the wound site moist. It is equally important that no regenerative tissue is peeled off on the removal of the dressing. 2-Methacryloyloxyethyl phosphorylcholine (MPC) has a phospholipid polar group that mimics a biomembrane. We prepared poly [MPC-co-n-dodecyl methacrylate (DMA)] (PMD), using conventional radical polymerization with 2,2'-azobisisobutyronitrile as an initiator, and coated it on polyurethane (PU; Tecoflex 60 Thermedics Inc.) membrane. Full-thickness surgical wounds were made on the dorsal skin of rats and wound healing was compared under the following three conditions: air-exposed control (no dressing), PU dressing, and PMD dressing. At 3, 4 and 7 days after the operation, the wound sizes of the PMD dressings were smaller than the non-dressed wound, and at 6 and 7 days after the operation, the wound sizes of PU dressing were smaller than that of the air-exposed group. But there were no significant difference between the PMD dressing group and PU dressing group. Histologically, scab formation was not observed on the PU or PMD-dressed wounds. However, in the air-exposed control, a scab was formed and re-epithelialization of the wound site was prevented. Additionally, no damage was observed in the histological section of PMD dressed wound after the wound was cured. These results indicate that PMD dressing (PMD-coated PU membrane) has the potential to provide an inert environment for wound healing as well as PU.

It has been reported that statin, an inhibitor of cholesterol synthesis, enhances bone formation by stimulating osteoblastic differentiation and function and that it also inhibits bone resorption by osteoclasts. Although the potentiality of statin as a therapeutic drug for osteoporosis has been expected, the clinical evidences supporting this potentiality are few. On the other hand, there are several reports demonstrating that local application of statin promotes bone augmentation and bone regeneration. The history of statin as a therapeutic drug for hypercholesterolemia is long and the toxicity of this drug has been well examined. Thus, it is unlikely that local application of this drug at low dose will induce serious side effects. It is possible that local bone augmentation with statin will appear clinically in the near future if proper local drug delivery system for statin is developed.

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

Apoptosis is known to be associated with wound healing and regeneration of dental pulp. We examined the effects of heat stress on clonal dental pulp cell line (RPC-C2A cells) to clarify the pulp wound healing process. RPC-C2A cells were exposed to heat stress at 43 degrees C for 45 min. After several time intervals, the inhibition of cell proliferation and apoptosis induction were analyzed by cell viability assay, DNA gel electrophoresis, nuclear staining, and terminal deoxynucleotidyl transferase mediated labeling assay. RPC-C2A cells showed the thermotolerance following heat stress. We found that apoptosis was induced in some RPC-C2A cells, whereas others remained alive, and observed the engulfment of apoptotic cells by scavenger-like RPC-C2A cells following heat stress. We also analyzed the phagocytotic activity of RPC-C2A cells and found that they had an ability to engulf apoptotic RPC-C2A cells, which was stimulated by heat stress. These results suggest that heat stress induces apoptosis of RPC-C2A cells, which are phagocytosed by the surviving RPC-C2A cells.

The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells. Introduction: We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system. Materials and Methods: Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM β-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture. Results: PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway. Conclusion: These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.

Objective: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. Materials and Methods: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2′-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. Results: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. Conclusion: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.

Expanding cells ex vivo is an important step in tissue engineering and the culture medium is usually supplemented with bovine serum. When a patient receives bone marrow stromal cells (BMSCs) grown in a medium containing bovine serum, infection of bovine diseases and/or the patient's unfavorable immune reaction to bovine proteins are of concern. To overcome these problems, we examined whether a patient's autologous serum could support the growth and differentiation of his/her BMSCs.Bone marrow was collected from the iliac by aspiration and cultured in a medium supplemented with 10% autologous serum or 10% fetal bovine serum (FBS). Number and area of the colonies of fibroblast-like cells (colony-forming unit fibroblast, CFU-f) were measured 8 days after the cell inoculation (day 8). Number and area of the alkaline phosphatase (ALP) positive colonies were measured on day 10. On day 40, the cultures were stained with alizarin red S. RNA was prepared from the culture on day 20, and the mRNA expression of osteoblastic genes was examined by reverse transcription polymerase chain reaction (RT-PCR) analysis.BMSCs, which were cultured in the medium supplemented with autologous serum, produced CFU-f, ALP-positive area and mineralized nodules, which is comparable to the BMSCs in the culture supplemented with FBS. The mRNA expressions of osteopontin, parathyroid hormone receptor, osteocalcin, and bone sialoprotein were detected in the culture supplemented with autologous serum.A patient's autologous serum could expand BMSCs without losing their potentiality for osteoblastic differentiation. Patients' autologous serum could be efficient to expand BMSCs and to be utilized safely for bone regeneration therapy.

The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system.

The effects of shark skin collagen on bone mineral density (BMD) were investigated in ovariectomized rats to breed by low protein foods. The rise of BMD was recognized to intake of collagen for two weeks. In the collagen group, the amount of collagen and proteoglycan in the epiphyses were increased. Shark skin collagen would be useful for as a food supplement for treating osteoporosis.

Few options for functional recovery using residual dental implants are available after removal of disintegrated implants. This report describes the application of a maxillary implant-supported removable partial denture (RPD) retained by a unilateral milled-bar-magnetic attachment combination fixed to the remaining two implants as an alternative for functional recovery without surgery. A custom milled-bar with keepers was fixed to the two remaining unparallel unilateral implants in a 78-year-old woman who visited our department after dental implant treatment. Although 2 of the 4 initial implants were lost, additional surgery was unfeasible; thus, a magnetic RPD with a clasp on the ipsilateral second molar was placed. Peri-implant infection and bone resorption did not occur in the 4 years after implant-supported RPD placement. In the present case, a custom milled-bar-magnetic attachment and a clasp combination have been effective for retaining an implant-supported RPD even with an inadequate unilateral design.

Bone morphogenetic protein family members (BMPs) are essential signaling molecules during limb development and, in this process, fibroblast growth factor family members (FGFs) cooperate with BMPs. FGFs also exert anabolic effects in bone when systemically or locally applied. Thus, it is likely that the cooperation with FGFs also occurs in BMP-induced ectopic bone formation and that the exogenous FGF application would promote this bone formation. In the present study, after subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on bone formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by fibroblastic mesenchymal cells; on day 7, cartilage tissue appeared; on day 10, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days 10, 14, and 21), whereas FGFRs were expressed continuously. In situ hybridization revealed that, on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day 10, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, 10, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced bone formation. We further suggest that the combination of rhBMP-2 and rhFGF-4 would be useful for bone augmentation.

The changes of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate (PR) during implant surgery under local anesthesia were analyzed in 38 normal healthy patients. The results were as follows: 1. SBP significantly increased compared with the preoperative control throughout surgery. 2. DBP did not change significantly compared with the preoperative control throughout surgery. 3. PR increased just after the local anesthesia, and lowered afterward. 4. Both SBP and DBP increased in correlation with age. 5. SBP, DBP, and PR increased in correlation with surgical procedure time. 6. SBP, DBP, and PR increased in correlation with number of implantation. From these results, it was indicated that implant surgery might be accompanied by a risk, even in healthy patients.

We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxyapatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17 beta-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 mumol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 mumol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6 is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

We have developed a novel osteotropic prodrug of estradiol (E(2)) conjugated with L-Asp-hexapeptide (E(2).3D(6)), which has very low affinity for estrogen receptors, and in this study, we examined its pharmacokinetic behavior and pharmacological potential. After a single iv injection of E(2) x 3D(6) to mice, the half-time for elimination from plasma was about 100 min; however, E(2) was selectively delivered to the bone and eliminated very slowly, declining to the endogenous level at about 7 days. After a single iv injection of E(2), the half-time in plasma was about 70 min, whereas E(2) was highly distributed to the uterus, and the bone concentration of E(2) was only slightly increased at 6 h. When E(2) (0.37 micromol/kg, sc, every third day) or E(2) x 3D(6) (0.11 to 1.1 micromol/kg, sc, every seventh day) was administered to OVX mice for 4 weeks, E(2) increased the bone mineral density (BMD) together with weights of liver and uterus, whereas E(2) x 3D(6) increased only the BMD, in a dose-dependent manner. E(2) x 3D(6) enhanced the expression of messenger RNAs of bone matrix proteins (osteopontin, bone sialoprotein, type I collagen alpha) of OVX mice at 4 h after administration, but E(2) did very slightly. These results indicate that the E(2) prodrug was delivered to the bone, where it gradually released E(2), thereby ameliorating bone loss. This acidic oligopeptide appears to be a good candidate for selective drug delivery to bone.

Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone, because HA, an inorganic component in hard tissues (bone and teeth), does not exist in soft tissues. Several bone noncollagenous proteins, which bind to HA, have repeating sequences of acidic amino acids in their structures as possible HA-binding sites. Thus, we think that a small peptide of repetitive acidic amino acid could work as a carrier for selective drug delivery to the bone. To test this hypothesis, we conjugated (Asp)6 to fluorescein isothiocyanate (FITC), evaluated its affinity to HA in vitro, and examined its tissue distribution after injection into rats. Although fluorescein itself did not bind to HA, (Asp)6-FITC bound to HA as well as calceine and tetracycline. Twenty-four hours after intravenous injection of (Asp)6-FITC to rats, animals were killed, and ground sections of hard tissues and cryosections of soft tissues were made. Under a confocal laser scanning microscope, clear labeling lines were observed in bones and teeth, whereas no labeling was detected in soft tissues. In the rats administered with fluorescein alone, the fluorescent labeling was detected in neither hard nor soft tissues. Fluorescent analysis of blood, urine, and bones after (Asp)6-FITC administration revealed that biological half-life of FITC in blood was short (60 minutes) and that within 24 h, 95% of the administered FITC was excreted as urine whereas 2% of the FITC accumulated in bones. After subcutaneous administration of (Asp)6-FITC to mice, fluorescent intensity remaining in the femurs was measured periodically. In these mice the biological half-life of FITC in the femur was 14 days. Present results indicate that (Asp)6 is effective as a carrier for selective drug delivery to bone.

Low-calcium-fed animals have been accepted as one of the experimental models showing a reduction in bone mass. However, the effects of short-term low-calcium feeding on bone turnover, the development of osteoprogenitor cells, and gene expression of bone matrix proteins have not been reported. In this study, we examined the effect of a low-calcium diet on rat tibia and analyzed the changes in the bone by histomorphometry, bone marrow cell culture, and in situ and Northern hybridization of the bone matrix proteins. Rats were fed either a low-calcium diet (0.05% Ca) or a normal calcium diet (0.5% Ca) using the pair feeding technique. They were killed at day 0, 12 h, and days 1, 2, and 3. In the low-calcium group, the serum parathyroid hormone (PTH) level was temporarily increased in 12 h after feeding the low-calcium diet. Bone mineral density in the trabecular bone was significantly decreased from 1 day after the low-calcium feeding, but cortical bone did not show any changes during the experimental period. The bone volume per tissue volume in the proximal tibia also decreased from day 1 in the low-calcium group. The number of osteoclasts and osteoblasts on the trabecular bone surface was increased in the low-calcium group compared with the normal-calcium group. An ex vivo study showed that the number of progenitors of osteoclasts and osteoblasts in bone marrow was also increased in the low-calcium group of rats. The localization of type I collagen mRNA was observed in osteoblasts in the low-calcium group. The Northern hybridization study showed that the gene expression of type I collagen, osteopontin, and osteocalcin was increased at day 3 in the low-calcium group. These results indicated that the trabecular bone surface quickly responded to the low-calcium feeding and that bone remodeling activity was activated probably by PTH. The changes in bone marrow cell populations and the gene expression of bone matrix proteins are closely associated with increased bone turnover induced by the low-calcium diet, resulting in rapid bone loss of the trabecular bone.

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis. Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration. We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4. Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5' end using the ExoIII system. Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector. Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified. The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect. Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4. DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis. These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone.

When Walker 256/S carcinosarcoma (W256/S) was subcutaneously inoculated into the back of mature female Wistar Imamichi rats (10-week-old), the tumor grew rapidly and caused increases in the urinary excretions of calcium and hydroxyproline, without changes in the serum concentrations of calcium and inorganic phosphorus. Furthermore, osteoporosis-like changes in the femurs and decrease in uterus weight were observed, as previously reported for W256/S-bearing young rats. In the healthy mature female rats, the estrus cycle passed through four stages (proestrus, estrus, metestrus and diestrus) within 4 to 5 days, with a peak of serum estradiol and progesterone levels in the proestrus stage. On the other hand, after subcutaneous inoculation of W256/S into the rats, the estrus cycle tended to pause upon the metestrus or diestrus stage, accompanied with significantly low estradiol and progesterone levels in serum. W256/S tumor produced and secreted luteinizing hormone-releasing hormone (LH-RH). In conclusion, it seems that the ectopical secretion of LH-RH from the tumor resulted in the decrease in the secretion of gonadotropic hormones, following low level of sex hormones and stopping the estrus cycle. Therefore, W256/S-bearing rats may be a model for osteoporosis of hypoovarianism or postmenopause.

We have reported that denbufylline, a phosphodiesterase 4 (PDE4) inhibitor, inhibits bone loss in Walker256/S tumor-bearing rats, suggesting therapeutic potentiality of a PDE4 inhibitor in osteopenia. In the present study, effects of a new PDE4 inhibitor, 1-n-butyl-3-n-propylxanthine (XT-44), in bone were evaluated in cell cultures and animal experiments. In rat bone marrow culture, XT-44 stimulated mineralized-nodule formation, whereas it inhibited osteoclast-like cell formation in mouse bone marrow culture. In Walker256/S-bearing rats (6-week-old female Wistar Imamichi rats), rapid decrease in bone mineral density (BMD) was prominent, and oral administration of XT-44 (0.3 mg/kg, every 2 days) inhibited the decrease in BMD. In the second animal experiment, female Wistar rats (6-week-old) were sciatic neurectomized, and XT-44 was orally administered to these rats every 2 days for 4 weeks. XT-44 administration (0.3 mg/kg) recovered BMD in these neurectomized animals. Furthermore, 19-week-old, female Wistar rats were ovariectomized (OVX), and 15 weeks after surgery, these rats were orally administered XT-44 every 2 days for 8 weeks. XT-44 treatment (1 mg/kg) increased the BMD of OVX rats. These results indicate that XT-44 could be a candidate as a therapeutic drug for treating osteopenia including osteoporosis.

Expression of parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor is one of the osteoblastic phenotypes; however, it has not been clear whether this phenotype expression is a marker of immature or mature osteoblasts. We examined the temporal expression pattern of PTH/PTHrP receptor in bone development in vivo and in vitro compared with the expression of other osteoblastic phenotypes: osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), alkaline phosphatase (ALP), and mineralization. Total RNA was extracted from rat calvariae, and cell culture of rat bone marrow at different developmental stages and then Northern blot hybridization were performed. Mineralization was detected with contact microradiography (CMR) in calvaria or with Alizarin Red S staining in bone marrow cell culture. Both in calvaria and in marrow cell culture, extensive expression of OPN, BSP, type I collagen (COL I), and ALP coincided with the onset of mineralization, and OC expression was observed after mineralized tissue formation. Notably, PTH/PTHrP receptor was expressed at an early developmental stage (prenatal day 14 in calvaria, day 5 in culture) when mineralized tissue was not formed and other osteoblastic phenotypes were scarcely detected. Further study in cell culture revealed that the fold increase in cyclic adenosine monophosphate (cAMP) in response to PTH was elevated with the advance in the culture stage. These results indicate that mRNA expression of PTH/PTHrP receptor could be the early differentiation marker in osteoblastic lineage and that the levels of cAMP production in response to PTH represent the stage of osteoblastic differentiation.

To reverse the adverse reactions of alkylxanthines and to develop novel inhibitors of cyclic AMP-specific phosphodiesterase (PDE IV), a series of heterocycle-condensed purines were designed and synthesized. Some of these new compounds had similar or more potent and selective inhibitory activity against PDE IV than known PDE IV inhibitors. The tracheal-relaxant activity of these compounds was closely correlated with their PDE IV-inhibitory activity. Moreover, these purine analogues did not have any positive-chronotropic action or adenosine-antagonistic action on isolated heart preparations, which are the particular adverse reactions of alkylxanthines. Among them, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (1c), which was the most selective and potent PDE IV inhibitor, did not cause emesis in Suncus murinus at a dosage range of 10-100 mg/kg (po), while an imidazole analogue of 1c (4c) and known PDE IV inhibitors such as rolipram and denbufylline caused emesis even at 10 or 30 mg/kg.

The effects of denbufylline, a xanthine derivative with selective inhibitory activity on the phosphodiesterase (PDE) 4 isoenzyme, on bone loss in Walker 256/S-bearing rats and on mineralized nodule formation and osteoclastlike cell formation in bone marrow culture systems were examined. Serial oral administrations of denbufylline inhibited the decrease in the bone mineral density of femurs from Walker 256/S-bearing rats, without influence on the healthy rats. Denbufylline restored the bone mass and the number of osteoclasts and osteoblasts per trabecular surface in the femur metaphysis. Among PDE inhibitors, only PDE4-selective inhibitors increased the number of mineralized nodules and decreased the number of osteoclastlike cells in the in vitro bone marrow culture systems, and dibutyryl cyclic AMP mimicked these effects in the in vitro systems. These results suggest that the PDE4 isoenzyme may play an important role in bone turnover through cyclic AMP and that its inhibitors are candidates for therapeutic drugs for the bone loss diseases.

Osteopontin (OPN) is a prominent bone matrix protein that is synthesized by osteoblastic cells. To elucidate the function of OPN in bone we studied the regulated expression of the rat OPN protein during bone formation in vivo and in vitro. OPN mRNA is expressed by preosteoblastic cells early in bone formation, but the highest expression is observed in mature osteoblasts at sites of bone remodelling. A low-phosphorylated, 55-kDa form of OPN is produced by the preosteoblastic cells, whereas osteoblasts produce a highly phosphorylated, 44-kDa protein; the two forms of OPN corresponding to pp69 and pp62 in transformed rat cells. The synthesis of the 55-kDa OPN correlates with the formation of a 'cement' matrix that is synthesized prior to bone deposition, whereas the 44-kDa OPN synthesized by osteoblasts associates rapidly with hydroxyapatite, possibly regulating crystal growth, and may also provide a substratum for osteoclast attachment. Expression of OPN mRNA is upregulated by growth and differentiation factors (PDGF, EGF, TGF-beta and BMP-7/OP-1) and by mechanical stress, which promote bone formation, as well as by osteotropic hormones (retinoic acid and vitamin D3), which can promote bone resorption and remodelling. However, OPN mRNA is down-regulated by bisphosphonates, which abrogate bone resorption. Regulation of OPN expression is, therefore, consistent with a multiplicity of functions for OPN that involve specific structural motifs in both the synthesis and resorption of bone.

Pulp tissue was obtained from maxillary incisors of young adult male Wistar rats, minced and digested with 0.5% trypsin and 0.02% EGTA at 37 degrees C for 30 min. Dissociated cells were cultured with or without 10 nM dexamethasone using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. Confluent cells were subcultured at 7 days and the medium further supplemented with beta-glycerophosphate (beta-GP). Dexamethasone in primary culture and/or secondary culture enhanced the formation of mineralized tissue while5 mM beta-GP was necessary for mineralization to occur. Biochemical analysis of the radiolabelled medium revealed that these cells produced type I, type I trimer and type III collagens. Analysis of [32PO4]-labelled medium, using DEAE-Sephacel ion-exchange chromatography and sodium dodecylsulphate-polyacrylamide gel electrophoresis, showed that these cells produced phosphophoryn-like protein. These results indicate that some of the rat dental pulp cells in culture express an odontoblast-like phenotype.

Confluent cultures of rat bone cells synthesize several forms of secreted phosphoprotein 1 (SPP-1, osteopontin), the major phosphorylated forms of which migrate at 55 and 44 kDa on 15% cross-linked SDS-PAGE gels and correspond to the transformation-associated proteins pp 69 and pp 62. A clonal rat calvarial cell line (RCA 11), which expressed the highest level of SPP-1, produced only the 55 kDa form of the phosphorylated protein, whereas normal rat calvarial cells enriched in osteoblastic cells (RC IV cells) produced mostly the 55 kDa form, with small amounts of the 44 kDa form. In contrast, a 44 kDa form was the major [32PO4]-labelled SPP-1 synthesized by a rat osteocarcoma cell line (ROS 17/2.8 cells) with lesser amounts of the 55 kDa SPP-1. When [35S]methionine was used to measure protein synthesis, only the 55 kDa SPP-1 could be clearly detected in confluent cultures of each cell population, indicating that the 55 kDa SPP-1 is the prominent form of SPP-1 synthesized by each cell population. Following treatment of the normal rat bone cells for 24 h with osteotropic hormones (vit D3, PTH and RA), growth factors (PDGF, EGF, TGF-beta), a tumor promoter (TPA) and a plant lectin (Con A), the 55 kDa [35S]methionine labelled SPP-1 was increased 1.7-8.3-fold. Similar, but generally lower responses were observed in the clonal RCA 11 cell line, whereas the ROS 17/2.8 cells were more refractory, showing only a strong response to vit D3. In general, vit D3 produced the strongest stimulation in all populations with TGF-beta producing a good response in the non-transformed cells and RA in the RC IV cells. In contrast, PTH was inhibitory in both RCA 11 and ROS 17/2.8 cells. In most, but not all, cases the alteration in SPP-1 synthesis reflected similar changes in SPP-1 mRNA and in the intensity of the [32PO4]-labelled 55 kDa SPP-1. Collectively, these studies demonstrate that bone cells produce several forms of SPP-1 which are differentially regulated in normal and transformed cells through both transcriptional and posttranscriptional mechanisms.

The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and beta-glycerophosphate (beta-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phosphatase was stimulated 6-, 5-, 3-, and 2.5-told, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-beta-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+ beta-GP). Extraction of the tissue matrix with 4 M GuHCl and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.